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Peng Huang,* Pengfei Rong, Albert Jin, Xuefeng Yan, Molly Gu Zhang, Jing Lin, Hao Hu, Zhe Wang, Xuyi Yue, Wanwan Li, Gang Niu, Wenbin Zeng, Wei Wang, Kechao Zhou, and Xiaoyuan Chen* Activatable theranostics are novel clinical solutions for early recognition, diagnosis, monitoring, and prognosis of diseases by enhancing contrast in various imaging modalities followed by tailored therapy.[1] Light, typically a wavelength-selected laser, as an exogenous stimulus with the advantage of spatiotemporal selectivity, has been extensively employed for photothermal, photodynamic, and/or photo-triggered chemo/gene therapy.[2] Photothermal therapy (PTT), which employs photothermal conversion agents (PTCAs) to destroy cancer tissues/cells by absorbing and transferring optical laser irradiation energy into heat, has been increasingly recognized as a promising minimally invasive alternative to traditional cancer treatments.[2d,3] Owing to the relatively low absorption/scattering of skin, tissue, blood, and water in the transparency window (650–950 nm), near-infrared (NIR) radiation–mediated PTCAs have shown effectiveness in in vivo PTT treatment.[4] A variety of organic/inorganic compounds/nanomaterials, such as indocyanine green (ICG), polyaniline nanoparticles (NPs), polypyrrole NPs, metal NPs (Au, Ag, Pd, and Ge),
Dr. P. Huang,[+] P. F. Rong,[+] X. F. Yan, M. G. Zhang, Dr. J. Lin, H. Hu, Dr. Z. Wang, Dr. X. Y. Yue, Prof. W. W. Li, Dr. G. Niu, Prof. X. Y. Chen Laboratory of Molecular Imaging and Nanomedicine (LOMIN) National Institute of Biomedical Imaging and Bioengineering (NIBIB) National Institutes of Health Bethesda, MD 20892, USA E-mail: [email protected]; [email protected] P. F. Rong, Prof. W. B. Zeng, Prof. K. C. Zhou State Key Laboratory for Powder Metallurgy Central South University Hunan, Changsha 410083, P. R. China P. F. Rong, Prof. W. Wang Department of Radiology The Third Xiangya Hospital Central South University Changsha, Hunan 410013, P. R. China Dr. A. Jin Laboratory of Cellular Imaging and Macromolecular Biophysics National Institute of Biomedical Imaging and Bioengineering (NIBIB) National Institutes of Health Bethesda, MD 20892, USA Dr. Z. Wang, Dr. X. Y. Yue Center for Molecular Imaging and Translational Medicine School of Public Health, Xiamen University Xiamen, Fujian 361005, P. R. China [+]These authors contributed equally to this work.
DOI: 10.1002/adma.201400914
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Dye-Loaded Ferritin Nanocages for Multimodal Imaging and Photothermal Therapy
semiconductor NPs (Cu2–xSe and CuS), and carbon-based materials (carbon nanotubes and graphenes), have been widely explored as PTCAs for PTT.[2a,5] However, most of these PTCAs are non-biodegradable, immunogenic, or have poor pharmacokinetics or potential long-term toxicity, which limits their clinical use.[3b,6] Therefore, development of novel biodegradable PTCAs with negligible toxicity and immunogenicity, suitable pharmacokinetics, high tumor accumulation rate, and real-time visualization is highly desirable to improve the in vivo PTT of cancer. Indocyanine green (ICG), a US Food and Drug Administration (FDA)-approved NIR dye, is not an effective PTT agent, owing to its serious photobleaching, short bloodstream circulation half-life, and low tumor accumulation rate.[7] Recently, NIR dye-loaded micelles have been reported to overcome several of the above-mentioned shortcomings for both NIR fluorescence imaging and PTT.[8] Due to the fact that both fluorescence imaging and phototherapy are strongly dependent on the light excitation wavelength, high fluorescence quantum yield (QY) of the dye is required for fluorescence imaging, at the expense of lowering the photothermal conversion efficiency for PTT. Therefore, achieving a balance between the dual roles of imaging contrast and photothermal conversion for optical imaging on the one hand and PTT on the other is a great challenge. Recently, several naturally occurring protein macromolecules have emerged as a novel class of drug delivery vehicles.[9] For example, albumin-based drug delivery systems such as methotrexate-albumin conjugates, an albumin-binding prodrug of doxorubicin (DOX), and nanoparticle albumin–bound paclitaxel (Abraxane) have been evaluated clinic.[9] Ferritin (FRT), a major iron storage protein in humans and most living organisms, has also been applied for efficient drug delivery.[10] The FRT nanocage is composed of 24 subunits, which selfassemble into a cage-like nanostructure with external and internal diameters of 12 and 8 nm, respectively. In nature, the internal cavity is suitable for loading different species.[11] We have demonstrated that doxorubicin precomplexed with Cu(II) can be loaded onto arginine-glycine-aspartate (RGD)-modified apoferritin nanocages with high efficiency, and zinc hexadecafluorophthalocyanine (ZnF16Pc) can be conveniently encapsulated into RGD4C-modified apoferritins (RFRTs).[10] FRT and its derivatives show great potential as a new type of drug carrier with high loading rates, good tumor selectivity, and small particle size. More importantly, FRT, being a natural polymer, raises fewer concerns for clinical translation than synthetic carriers.
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Figure 1. a) Topological image of FRT in partial layer coverage on a mica surface with 40 nm color scale for the height. b) Selected FRT nanocages are presented on a 20 nm grid in 3D to visualize protein coats with color scale from 8–12 nm in the inset. c) A representative AFM image of DFRT on a mica surface with 80 nm color scale for the height. d) Section plot along the blue line in (a) and white line in (c), showing DFRT particles are larger and more variable in size. e) Histogram distribution of the equivalent spherical (Eq.) diameter (Eq. diameter = (6V/π)1/3) for the FRT and DFRT nanoparticles, showing significant loading of the dye. Inset: 3D visualization of DFRT nanoparticles placed on a 100 nm grid.
In this Communication, we report for the first time a novel “chameleon” theranostic platform based on NIR dye (new cyanine green, IR820)–loaded ferritin (DFRT) nanocages with strong absorbance in the NIR region, due to the strong interaction between IR820 dye and FRT, for photoacoustic/fluorescence multimodal imaging-guided PTT.[12,13] With two different excitation wavelengths, 550 nm for fluorescence imaging with high fluorescence QY and 808 nm for photoacoustic imaging (PAI) and PTT with high photothermal conversion efficiency, we successfully achieved simultaneous optimization of dual roles between imaging contrast and photothermal conversion for imaging-guided PTT. DFRT was formed by “opening” and “closing” the FRT nanocages in the presence of dye molecules. The dye encapsulation was carried out using direct and step-wise change of pH of the solutions from 2 to 7.4.[10,14] Both FRT and DFRT were characterized similarly by atomic force microscopy (AFM) (Figure 1). Figure 1a,b shows the AFM image of the original FRT as well-defined spherical nanoparticles. The 3D AFM image in Figure 1b clearly visualizes the protein coats on the
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surface of FRT. DFRT appears to be slightly larger and more variable in size than FRT (Figure 1c). As shown in Figure 1d, the diameter of FRT is around 12 nm (determined by AFM measured height), matching the theoretical value, with slight broadening in the X–Y direction due to nanoparticle compression and AFM-tip size. The histogram distributions of the equivalent spherical diameter, (6V/π)1/3, for the FRT and DFRT nanoparticles shown in Figure 1e indicate significant loading of IR820 dye molecules. Next we investigated the photophysical and photochemical properties of DFRT using optical absorption and fluorescence spectra. Figure 2a and Figure S1 (Supporting Information) show UV-vis absorbance spectra of FRT, IR820 dye, and DFRT. The IR820 dye exhibits a strong peak at 687 nm, and a weak shoulder at 825 nm. FRT without dye shows virtually no absorption in the range 500–900 nm. After the dye is loaded into FRT, DFRT exhibits a weak shoulder at 761 nm, and a strong peak at 835 nm. The remarkably red-shifted absorption may be attributed to the strong interaction between the dye and FRT. The loading process is accompanied by a color
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COMMUNICATION Figure 2. Characterization of DFRT. a) UV-vis absorbance spectra of FRT, IR820 dye, and DFRT. b) Fluorescence emission spectra of IR820 dye and DFRT. c) Temperature elevation of aqueous solution samples with the same concentration as a function of irradiation time. d) Photoacoustic (PA) spectrum of DFRT.
change of the solution from dark green to brown (Figure S2a, Supporting Information). The absorption spectrum of DFRT is different from that of unloaded IR820 dye (Figure S2b–d). With different mole ratios of ferritin and IR820, that is, 1:50, 1:80, and 1:100, the IR820 loading efficiencies were 17.32, 25.11, and 29.53 wt%, respectively. We chose a mole ratio of 1:50 for the remaining experiments. The fluorescence excitation spectrum of IR820 dye shows the two excitation wavelengths, at 550 and 770 nm (Figure S3, Supporting Information). As shown in Figure 2b, both IR820 dye and DFRT exhibited a shift in emission peak from 604 nm to 834 nm as the excitation wavelength was changed from 550 nm to 770 nm. The results also show fluorescence quenching, most likely owing to the tight packing of IR820 dye molecules after being loaded into FRT. The quenching efficiency of IR820 dye in DFRT is ca. 20% upon 550 nm excitation, but almost ca. 80% upon 770 nm excitation. Based on these results, we selected 550 nm for fluorescence imaging with high fluorescence QY and 808 nm for PTT with high photothermal conversion efficiency. When separate excitation wavelengths are used, it is easy to alternate between the dual roles of DFRT — contrast agent and PTCA — for imaging and PTT. To further verify the red-shifted absorption and fluorescence quenching of dye loaded FRT, we chose ICG, a FDA-approved NIR dye, for comparison. Similar results were observed, as shown in Figure S4 (Supporting Information). ICG has a strong peak at 683 nm, and a weak shoulder at 780 nm. After being
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encapsulated into FRT, ICG-FRT exhibits a weak shoulder at 725 nm, and a strong peak at 800 nm. The loading process is also accompanied by a color change of the solution from light green to black (Figure S2). A marked fluorescence quenching of ICG upon 760 nm excitation was found after FRT loading. Interestingly, there is ca. 4.5-fold fluorescence enhancement of ICG at the 700 nm emission peak upon 630 nm excitation in the ICG-FRT system. Therefore, ICG-FRT is also suitable for simultaneous optimization of imaging and PTT using separate wavelengths, 630 nm for fluorescence imaging with high fluorescence QY and 808 nm for PAI and PTT with high photothermal conversion efficiency. These results suggest the great potential of FRT as a new type of carrier for dye delivery, fluorescence, and PAI, and enhanced PTT treatment. We next studied the potential of DFRT in PTT of cancer by measuring the temperature elevation of FRT, IR820 dye, and DFRT in aqueous solutions exposed to a 1 W cm–2 808 nm laser for 5 min (Figure 2c). No obvious temperature change was observed in the control group of pure FRT, owing to the lack of absorption of FRT in the range 500–900 nm. Upon the same laser exposure, IR820 dye and DFRT (80 µg mL–1 of dye) exhibited a temperature rise of 18.34 and 28.38 °C, respectively. These results suggest significantly higher photothermal conversion efficiency of DFRT compared to the IR820 dye upon 808 nm excitation. The effective photothermal conversion also supports the use of DFRT for PAI[15] (Figure 2d). The best excitation wavelength range is from 800 to 850 nm. To achieve
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Figure 3. In vitro cell experiments. a) Fluorescence images of calcein AM / PI co-stained 4T1 cells after incubation for 6 h with free IR820 dye or DFRT (50 µg mL–1 dye) after being exposed to 808 nm laser at different power densities of 0.5 and 1 W cm–2 for 5 min. Cells incubated with the same concentration of free IR820 dye or DFRT without laser irradiation were chosen as controls. b) Viability of 4T1 cells after being incubated with various concentrations of DFRT for 24 h. c) Viabilities of 4T1 cells after DFRT-induced PTT at different laser power densities. Cell viability was normalized to the control group without any treatment. Error bars are based on the standard deviations of five parallel samples.
in vivo simultaneous PAI and PTT, we chose 808 nm as the excitation wavelength for PAI. Encouraged by high photothermal conversion efficiency of DFRT upon 808 nm excitation, we next investigated the in vitro PTT efficacy and cytotoxicity of DFRT. Live/dead cells were differentiated by calcein AM (live cells, green fluorescence) and propidium iodide (PI) (dead cells, red fluorescence) co-staining after PTT treatment (Figure 3a). In the control groups (IR820 dye or DFRT only), all the cells displayed green fluorescence, so IR820 dye or DFRT has no measurable cytotoxicity. Upon laser irradiation at different power densities, both IR820 dye and DFRT show a laser dose–dependent PTT effect on 4T1 cells. All cells were killed after being incubated with DFRT (50 µg mL–1 of IR820 dye) and exposed to the NIR laser at 1 W cm–2 for 5 min. As shown in Figure S5 (Supporting Information), on the boundary of the laser spot, only cells within the laser spot were killed, showing intense homogeneous red fluorescence. The cells outside the region of the laser spot stayed alive, showing strong green fluorescence. To further evaluate the cytotoxicity and PTT efficacy of DFRT, the viability of 4T1 cells after being exposed to various concentrations of DFRT for 24 h without laser irradiation were assessed (Figure 3b). We found DFRT exhibited negligible toxicity at all the test concentrations. Even with 400 µg mL–1 of DFRT, the cell viability is over 90%, which suggests that DFRT alone has negligible adverse effect on tumor cells. To verify the improved PTT efficacy induced by DFRT, three groups — with FRT, IR820 dye, or DFRT — were compared under the same conditions with increasing laser power. For the FRT group, no PTT effect was observed at any of the studied laser power densities. Upon laser irradiation, both IR820 dye and DFRT
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induced a laser dose–dependent cytotoxicity to 4T1 cells, which was significantly different from that of control groups as shown in Figure 3c. DFRT exhibited a much higher PTT efficacy than IR820 dye under the same conditions, which is attributed to the higher photothermal conversion efficiency of DFRT than IR820 dye. The above results are in good agreement with live/dead cell staining results. To investigate the stability of DFRT at the cellular level, we prepared fluoroscein isothiocyanate (FITC)-conjugated cyanine5.5 (Cy5.5)-loaded FRT (FITC-FRT-Cy5.5). FITC was covalently coupled to primary amines of FRT. FRT was loaded with Cy5.5 by the same method. After 6 h incubation of FITC-FRTCy5.5 with 4T1 cells, we used LysoTracker (a fluorescent dye probe) to stain lysosomes and observed FITC-FRT-Cy5.5 using confocal laser scanning microscopy (CLSM). Figure S6 (Supporting Information) clearly shows that the blue fluorescence signals from LysoTracker match well with the fluorescence signals from FITC-FRT (green) and Cy5.5 (red), suggesting FITCFRT and Cy5.5 colocalize within the lysosomes. These results indicate FITC-FRT-Cy5.5 is stable in lysosomes during the 6 h incubation. It would be reasonable to believe that DFRT is also stable in lysosomes. We next investigated the feasibility of using DFRT for in vivo multimodal imaging-guided PTT. To achieve the simultaneous optimization of NIR fluorescence and PAI, we used two separate wavelengths to excite DFRT: 550 nm for fluorescence imaging with high fluorescence QY and 808 nm for PAI with high photothermal conversion efficiency. Nude mice with subcutaneous 4T1 tumors were selected as the animal model. When the tumors reached 60 mm3, the mice were treated with an intravenous injection of DFRT (2 mg mL–1 of IR820 dye,
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COMMUNICATION Figure 4. In vivo NIR fluorescence (FL) and photoacoustic (PA) imaging. a)In vivo NIR FL images in mice taken at different times after intravenous injection of DFRT. b) Ex vivo NIR FL images of mouse tissues (from left to right: heart, liver, spleen, lung, kidneys, tumor), harvested 24 h after injection of free IR820 dye or DFRT. c)In vivo 2D and 3D cross-sectional US and PA images of tumor tissues in mice taken at different times after intravenous injection of free IR820 dye or DFRT. d) Change of FL signal of tumors with time following intravenous injection of DFRT. e) Biodistribution of free IR820 dye or DFRT in 4T1 tumor-bearing nude mice 24 h after injection. f) Change of PA signal of tumors with time following intravenous injection of free IR820 dye or DFRT. For all plots: mean ± standard deviation; n = 3 per group
200 µL). NIR fluorescence imaging was employed to monitor the pharmacokinetics of DFRT in vivo. As shown in Figure 4a, distributed DFRT fluorescence was found over the whole body soon after injection. With time, the whole-body signal gradually decreased and the tumor signal increased. The tumor can be easily differentiated from the surrounding normal tissue with good contrast 4–24 h post-injection. After 24 h, the major organs of mice injected with unloaded dye or DFRT were harvested for ex vivo imaging (Figure 4b). Meanwhile, PAI was also used to observe the PA signal change in tumor tissues. Figure 4c shows snapshots of mice taken at various times of in vivo two-dimensional (2D) and 3D cross-sectional ultrasonic (US) and PA images upon 808 nm excitation of the tumor area after intravenous injection of IR820 dye or DFRT. A gradual increase of PA signal was observed in the tumor regions of DFRT-treated mice. DFRT accumulation in the tumor was quantified by dye fluorescence intensity (Figure 4d). The average fluorescence intensity from dye accumulated in the tumor areas quickly rose in the period 1–4 h after injection and reached a plateau 4–24 h after injection, followed by a slight decrease over time. Meanwhile, the quantification of tissue/organ distribution was carried out by means of ex vivo imaging (Figure 4e). The relative
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uptake efficiency (10.48 ± 0.93%) of DFRT is ca. four times that of IR820 dye (2.64 ± 0.20%), which suggests that the FRT carrier significantly improved the tumor accumulation rate of the dye. The strong fluorescence signals in the liver and spleen may be attributed to the macrophage clearance of IR820 dye and DFRT from the blood. Strong kidney signals imply renal clearance of IR820 dye and DFRT. Semi-quantification of PA signals in the tumor area revealed that the average tumor PA intensity (0.95 ± 0.01 a.u.) of DFRT was ca. 2.6 times that of IR820 dye (0.37 ± 0.02 a.u.) (Figure 4f), which further confirms that the FRT carrier can significantly improve tumor accumulation of the dye molecules. The tumor tissue turned a dark color with increased accumulation of DFRT at long times after intravenous injection (Figure S7, Supporting Information). Based on the effective DFRT-induced PTT in vitro and the high tumor accumulation rate of DFRT in vivo, in vivo PTT studies were carried out on the same animal model. According to the observations of fluorescence and PA imaging, the most suitable time to implement PTT is 24 h after injection of DFRT. Upon 808 nm laser irradiation, thermal imaging was employed to monitor the temperature change in vivo using an infrared (IR) thermal camera (Figure 5a,c). For mice injected with DFRT, the tumor temperature changes were 23.8 or 42.2 °C
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Figure 5. In vivo PTT. a) Thermal images of 4T1 tumor-bearing mice after injection of free IR820 dye or DFRT and exposure to 808 nm laser irradiation. b) 3D color Doppler images of tumor before and 12 h after PTT treatment. c) Heating curves of 4T1 tumors upon laser irradiation as a function of irradiation time. d) Tumor growth curves of different groups of 4T1 tumor-bearing mice after treatment. Tumor volumes have been normalized to their initial sizes. Error bars represent the standard deviations of 4–6 mice per group. * P < 0.01. e) Survival curves of tumor-bearing mice after various treatments. DFRT-injected mice, after PTT treatment, showed 100% survival over 40 days. f) H&E staining of tumor sections collected from different groups of mice 2 h after laser irradiation.
within 10 min, upon 0.5 or 1 W cm–2 808 nm laser irradiation, respectively. No significant temperature rise was observed in other body parts of the mice (Figure S8, Supporting Information). In contrast, upon 1 W cm–2 808 nm laser irradiation, the local temperature of tumors treated with phosphate-buffered saline (PBS) or IR820 dye increased by about 7.2 or 11.1 °C, respectively. Figure 5b shows 3D color Doppler images of a tumor before and after PTT treatment. While the tumor clearly displayed blood flow prior to treatment, no blood flow was observed in the tumor after treatment, which can be attributed to the destruction of the blood vessels of tumor tissue during treatment. For in vivo DFRT-induced PTT treatment, eight groups of 4T1 tumor-bearing mice (4–6 mice per group) were designated
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as follows: untreated mice (control, n = 6), mice with PBS administration and laser irradiation (PBS+laser, n = 6), mice with FRT administration but no laser irradiation (FRT, n = 4), mice with FRT administration and laser irradiation (FRT+laser, n = 4), mice with dye administration but no laser irradiation (IR820 dye, n = 4), mice with dye administration and laser irradiation (IR820 dye+laser, n = 5), mice with DFRT administration but no laser irradiation (DFRT, n = 4), and mice with DFRT administration and laser irradiation (DFRT+laser, n = 6). The same 808 nm laser density (0.5 W cm–2) and exposure time (10 min) were applied in all eight treatments. While the tumors in the control groups all grew at similar speeds, the tumors in both the IR820 dye+laser and DFRT+laser groups exhibited remarkable delay in tumor growth or tumor regression after
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and 808 nm for PAI and PTT with high photothermal conversion efficiency, we achieved optimization of both imaging and PTT in our system. The cancer theranostic capability of DFRT was carefully investigated both in vitro and in vivo. Most intriguingly, 100% in vivo tumor elimination is produced by intravenous injection of DFRT, under a low laser power density of 0.5 W cm–2 without weight loss, tumor recurrence, and noticeable toxicity. This concept of protein-based theranostics has potential applications for clinical translation of personalized nanomedicine with targeted dye/drug delivery, diagnosis, and treatment.
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two weeks (IR820 dye+laser vs. ctrl, P < 0.05; DFRT+laser vs. ctrl, P < 0.001) (Figure 5d). In the DFRT+laser group, all the tumors were effectively ablated, leaving scars at their original sites, without showing recurrence (Figure S9, Supporting Information). Additionally, it is also worth noting that the DFRT group exhibited significantly higher PTT therapeutic efficacy than the IR820 dye+laser group on day 14 (DFRT+laser vs. IR820 dye+laser, P < 0.0001). As shown in Figure 5e, DFRT administration and laser irradiation greatly prolonged mice survival over 40 days, without a single death or tumor recurrence. In contrast, for the IR820 dye+laser group, all the mice had to be sacrificed on day 20 owing to the extensive tumor burden. Mice in other control groups showed average lifespans of ca. 14 days after treatment started. Hematoxylin and eosin (H&E) stains of tumor sections were collected from four laser irradiation groups of mice 2 h after laser irradiation (Figure 5f). For the PBS+laser and FRT+laser groups, the histological section showed observable errhysis (minor bleeding) and the infiltration of a small number of inflammatory cells without tissue structure damage. For the IR820 dye+laser group, inflammatory cell infiltration, cell death, and errhysis with partial tissue structure damage was observed. For the DFRT+laser group, similar results were observed; however, instead of errhysis with partial tissue structure damage, serious tissue structure damage was detected. In the high-resolution H&E images (Figure S10, Supporting Information), we found significant cancer cell damage with nuclear membrane fragmentation (karyorrhexis) and nuclei shrinkage with pyknosis (condensation of chromatin) in the DFRT+laser group. The irreversible condensation of chromatin and karyorrhexis in the nuclei of tumor cells led to intensive necrosis or apoptosis. The results indicated that the in vivo PTT therapeutic efficacy of DFRT was significantly higher than that of IR820 dye. Furthermore, no significant body weight variation was noticed after DFRT-induced PTT treatment (Figure S11, Supporting Information). After DFRT administration and laser irradiation, main organs from different groups of mice, as seen through H&E stained images (Figure S12, Supporting Information), showed no obvious damage or inflammation. In accordance with the fact that DFRT has low cytotoxicity and immunogenicity, it is valid to surmise that circulating DFRT does not induce discernible toxic side effects in treated animals. Thus, owing to its excellent theranostic capability without noticeable toxicity and immunogenicity, DFRT is a suitable candidate for multimodal imaging-guided PTT in vivo. The following may be considered in future applications of DFRT for cancer theranostics: 1) control of the arrangement of dye molecules inside FRT; 2) precise control of the size of DFRT; 3) enhanced knowledge of the stability of DFRT in vivo; 4) modification of the surface of FRT with specific biomarkers to further improve the tumor accumulation rate of DFRT; 5) systematic assessment of the toxicity, biocompability, immunogenicity, pharmacokinetics, and biodistribution of DFRT in vivo. In summary, we have developed a ferritin (DFRT) nanocage, loaded with the near-infrared dye IR820, that shows strong absorbance in the NIR region for simultaneous fluorescence/ photoacoustic/photothermal multimodal imaging-guided enhanced PTT. Using two different wavelengths for excitation, 550 nm for fluorescence imaging with high fluorescence QY
Supporting Information Supporting Information is available from the Wiley Online Library or from the authors.
Acknowledgements This work was supported by the National Key Basic Research Program (973 Project) (2010CB933901, 2013CB733802, 2014CB744503), the National Science Foundation of China (81272987, 31170961, 51102258, 81371596), and the Intramural Research Program (IRP) of the NIBIB, NIH. Received: February 26, 2014 Revised: June 24, 2014 Published online: August 14, 2014 [1] a) J. F. Lovell, C. S. Jin, E. Huynh, H. Jin, C. Kim, J. L. Rubinstein, W. C. W. Chan, W. Cao, L. V. Wang, G. Zheng, Nat. Mater. 2011, 10, 324–332; b) P. Rai, S. Mallidi, X. Zheng, R. Rahmanzadeh, Y. Mir, S. Elrington, A. Khurshid, T. Hasan, Adv. Drug Delivery Rev. 2010, 62, 1094–1124; c) P. Huang, J. Lin, X. Wang, Z. Wang, C. Zhang, M. He, K. Wang, F. Chen, Z. Li, G. Shen, D. Cui, X. Chen, Adv. Mater. 2012, 24, 5104–5110; d) J. F. Lovell, T. Liu, J. Chen, G. Zheng, Chem. Rev. 2010, 110, 2839–2857. [2] a) Z. Zhang, J. Wang, C. Chen, Adv. Mater. 2013, 25, 3869–3880; b) W. S. Kuo, C. N. Chang, Y. T. Chang, M. H. Yang, Y. H. Chien, S. J. Chen, C. S. Yeh, Angew. Chem. Int. Ed. 2010, 122, 2771–2775; c) P. Huang, C. Xu, J. Lin, C. Wang, X. Wang, C. Zhang, X. Zhou, S. Guo, D. Cui, Theranostics 2011, 1, 240–250; d) P. Huang, L. Bao, C. Zhang, J. Lin, T. Luo, D. Yang, M. He, Z. Li, G. Gao, B. Gao, S. Fu, D. Cui, Biomaterials 2011, 32, 9796–9809; e) J. Lin, S. Wang, P. Huang, Z. Wang, S. Chen, G. Niu, W. Li, J. He, D. Cui, G. Lu, X. Chen, Z. Nie, ACS Nano 2013, 7, 5320–5329; f) P. Huang, J. Lin, S. Wang, Z. Zhou, Z. Li, Z. Wang, C. Zhang, X. Yue, G. Niu, M. Yang, D. Cui, X. Chen, Biomaterials 2013, 34, 4643–4654; g) S. Wang, P. Huang, L. Nie, R. Xing, D. Liu, Z. Wang, J. Lin, S. Chen, G. Niu, G. Lu, X. Chen, Adv. Mater. 2013, 25, 3055–3061; h) B. Jang, Y. Choi, Theranostics 2012, 2, 190–197. [3] a) J. Zeng, D. Goldfeld, Y. Xia, Angew. Chem. Int. Ed. 2013, 52, 4169–4173; b) P. Huang, J. Lin, W. Li, P. Rong, Z. Wang, S. Wang, X. Wang, X. Sun, M. Aronova, G. Niu, R. D. Leapman, Z. Nie, X. Chen, Angew. Chem. Int. Ed. 2013, 52, 13958–13964. [4] a) Y. Wang, K. C. Black, H. Luehmann, W. Li, Y. Zhang, X. Cai, D. Wan, S.-Y. Liu, M. Li, P. Kim, ACS Nano 2013, 7, 2068–2077; b) K. Yang, H. Xu, L. Cheng, C. Sun, J. Wang, Z. Liu, Adv. Mater. 2012, 24, 5586–5592. [5] a) Z. Zha, J. Wang, E. Qu, S. Zhang, Y. Jin, S. Wang, Z. Dai, Sci. Rep. 2013, 3, doi: 10.1038/srep02360; b) Q. Tian, J. Hu, Y. Zhu, R. Zou,
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Z. Chen, S. Yang, R.-W. Li, Q. Su, Y. Han, X. Liu, J Am. Chem. Soc. 2013, 135, 8571–8577; c) Q. Tian, F. Jiang, R. Zou, Q. Liu, Z. Chen, M. Zhu, S. Yang, J. Wang, J. Wang, J. Hu, ACS Nano 2011, 5, 9761–9771; d) Q. Tian, M. Tang, Y. Sun, R. Zou, Z. Chen, M. Zhu, S. Yang, J. Wang, J. Wang, J. Hu, Adv. Mater. 2011, 23, 3542–3547; e) C. M. Hessel, V. P. Pattani, M. Rasch, M. G. Panthani, B. Koo, J. W. Tunnell, B. A. Korgel, Nano Lett. 2011, 11, 2560–2566; f) P. Huang, P. Rong, J. Lin, W. Li, X. Yan, M. Zhang, L. Nie, G. Niu, J. Lu, W. Wang, X. Chen, J. Am. Chem. Soc. 2014, 136, 8307–8313; g) H. Shen, L. Zhang, M. Liu, Z. Zhang, Theranostics 2012, 2, 283. [6] Z. Zha, Z. Deng, Y. Li, C. Li, J. Wang, S. Wang, E. Qu, Z. Dai, Nanoscale 2013, 5, 4462–4467. [7] a) Z. Zha, X. Yue, Q. Ren, Z. Dai, Adv. Mater. 2012, 25, 777–782; b) C. Zheng, M. Zheng, P. Gong, D. Jia, P. Zhang, B. Shi, Z. Sheng, Y. Ma, L. Cai, Biomaterials 2012, 33, 5603–5609. [8] a) M. Zheng, C. Yue, Y. Ma, P. Gong, P. Zhao, C. Zheng, Z. Sheng, P. Zhang, Z. Wang, L. Cai, ACS Nano 2013, 7, 2056–2067; b) C. Yue, P. Liu, M. Zheng, P. Zhao, Y. Wang, Y. Ma, L. Cai, Biomaterials 2013, 34, 6853–6861; c) L. Cheng, W. He, H. Gong, C. Wang, Q. Chen, Z. Cheng, Z. Liu, Adv. Funct. Mater. 2013, 23, 5893–5902; d) Z. Wan,
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Peng Huang,* Pengfei Rong, Albert Jin, Xuefeng Yan, Molly Gu Zhang, Jing Lin, Hao Hu, Zhe Wang, Xuyi Yue, Wanwan Li, Gang Niu, Wenbin Zeng, Wei Wang, Kechao Zhou, and Xiaoyuan Chen* Activatable theranostics are novel clinical solutions for early recognition, diagnosis, monitoring, and prognosis of diseases by enhancing contrast in various imaging modalities followed by tailored therapy.[1] Light, typically a wavelength-selected laser, as an exogenous stimulus with the advantage of spatiotemporal selectivity, has been extensively employed for photothermal, photodynamic, and/or photo-triggered chemo/gene therapy.[2] Photothermal therapy (PTT), which employs photothermal conversion agents (PTCAs) to destroy cancer tissues/cells by absorbing and transferring optical laser irradiation energy into heat, has been increasingly recognized as a promising minimally invasive alternative to traditional cancer treatments.[2d,3] Owing to the relatively low absorption/scattering of skin, tissue, blood, and water in the transparency window (650–950 nm), near-infrared (NIR) radiation–mediated PTCAs have shown effectiveness in in vivo PTT treatment.[4] A variety of organic/inorganic compounds/nanomaterials, such as indocyanine green (ICG), polyaniline nanoparticles (NPs), polypyrrole NPs, metal NPs (Au, Ag, Pd, and Ge),
Dr. P. Huang,[+] P. F. Rong,[+] X. F. Yan, M. G. Zhang, Dr. J. Lin, H. Hu, Dr. Z. Wang, Dr. X. Y. Yue, Prof. W. W. Li, Dr. G. Niu, Prof. X. Y. Chen Laboratory of Molecular Imaging and Nanomedicine (LOMIN) National Institute of Biomedical Imaging and Bioengineering (NIBIB) National Institutes of Health Bethesda, MD 20892, USA E-mail: [email protected]; [email protected] P. F. Rong, Prof. W. B. Zeng, Prof. K. C. Zhou State Key Laboratory for Powder Metallurgy Central South University Hunan, Changsha 410083, P. R. China P. F. Rong, Prof. W. Wang Department of Radiology The Third Xiangya Hospital Central South University Changsha, Hunan 410013, P. R. China Dr. A. Jin Laboratory of Cellular Imaging and Macromolecular Biophysics National Institute of Biomedical Imaging and Bioengineering (NIBIB) National Institutes of Health Bethesda, MD 20892, USA Dr. Z. Wang, Dr. X. Y. Yue Center for Molecular Imaging and Translational Medicine School of Public Health, Xiamen University Xiamen, Fujian 361005, P. R. China [+]These authors contributed equally to this work.
DOI: 10.1002/adma.201400914
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Dye-Loaded Ferritin Nanocages for Multimodal Imaging and Photothermal Therapy
semiconductor NPs (Cu2–xSe and CuS), and carbon-based materials (carbon nanotubes and graphenes), have been widely explored as PTCAs for PTT.[2a,5] However, most of these PTCAs are non-biodegradable, immunogenic, or have poor pharmacokinetics or potential long-term toxicity, which limits their clinical use.[3b,6] Therefore, development of novel biodegradable PTCAs with negligible toxicity and immunogenicity, suitable pharmacokinetics, high tumor accumulation rate, and real-time visualization is highly desirable to improve the in vivo PTT of cancer. Indocyanine green (ICG), a US Food and Drug Administration (FDA)-approved NIR dye, is not an effective PTT agent, owing to its serious photobleaching, short bloodstream circulation half-life, and low tumor accumulation rate.[7] Recently, NIR dye-loaded micelles have been reported to overcome several of the above-mentioned shortcomings for both NIR fluorescence imaging and PTT.[8] Due to the fact that both fluorescence imaging and phototherapy are strongly dependent on the light excitation wavelength, high fluorescence quantum yield (QY) of the dye is required for fluorescence imaging, at the expense of lowering the photothermal conversion efficiency for PTT. Therefore, achieving a balance between the dual roles of imaging contrast and photothermal conversion for optical imaging on the one hand and PTT on the other is a great challenge. Recently, several naturally occurring protein macromolecules have emerged as a novel class of drug delivery vehicles.[9] For example, albumin-based drug delivery systems such as methotrexate-albumin conjugates, an albumin-binding prodrug of doxorubicin (DOX), and nanoparticle albumin–bound paclitaxel (Abraxane) have been evaluated clinic.[9] Ferritin (FRT), a major iron storage protein in humans and most living organisms, has also been applied for efficient drug delivery.[10] The FRT nanocage is composed of 24 subunits, which selfassemble into a cage-like nanostructure with external and internal diameters of 12 and 8 nm, respectively. In nature, the internal cavity is suitable for loading different species.[11] We have demonstrated that doxorubicin precomplexed with Cu(II) can be loaded onto arginine-glycine-aspartate (RGD)-modified apoferritin nanocages with high efficiency, and zinc hexadecafluorophthalocyanine (ZnF16Pc) can be conveniently encapsulated into RGD4C-modified apoferritins (RFRTs).[10] FRT and its derivatives show great potential as a new type of drug carrier with high loading rates, good tumor selectivity, and small particle size. More importantly, FRT, being a natural polymer, raises fewer concerns for clinical translation than synthetic carriers.
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Figure 1. a) Topological image of FRT in partial layer coverage on a mica surface with 40 nm color scale for the height. b) Selected FRT nanocages are presented on a 20 nm grid in 3D to visualize protein coats with color scale from 8–12 nm in the inset. c) A representative AFM image of DFRT on a mica surface with 80 nm color scale for the height. d) Section plot along the blue line in (a) and white line in (c), showing DFRT particles are larger and more variable in size. e) Histogram distribution of the equivalent spherical (Eq.) diameter (Eq. diameter = (6V/π)1/3) for the FRT and DFRT nanoparticles, showing significant loading of the dye. Inset: 3D visualization of DFRT nanoparticles placed on a 100 nm grid.
In this Communication, we report for the first time a novel “chameleon” theranostic platform based on NIR dye (new cyanine green, IR820)–loaded ferritin (DFRT) nanocages with strong absorbance in the NIR region, due to the strong interaction between IR820 dye and FRT, for photoacoustic/fluorescence multimodal imaging-guided PTT.[12,13] With two different excitation wavelengths, 550 nm for fluorescence imaging with high fluorescence QY and 808 nm for photoacoustic imaging (PAI) and PTT with high photothermal conversion efficiency, we successfully achieved simultaneous optimization of dual roles between imaging contrast and photothermal conversion for imaging-guided PTT. DFRT was formed by “opening” and “closing” the FRT nanocages in the presence of dye molecules. The dye encapsulation was carried out using direct and step-wise change of pH of the solutions from 2 to 7.4.[10,14] Both FRT and DFRT were characterized similarly by atomic force microscopy (AFM) (Figure 1). Figure 1a,b shows the AFM image of the original FRT as well-defined spherical nanoparticles. The 3D AFM image in Figure 1b clearly visualizes the protein coats on the
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surface of FRT. DFRT appears to be slightly larger and more variable in size than FRT (Figure 1c). As shown in Figure 1d, the diameter of FRT is around 12 nm (determined by AFM measured height), matching the theoretical value, with slight broadening in the X–Y direction due to nanoparticle compression and AFM-tip size. The histogram distributions of the equivalent spherical diameter, (6V/π)1/3, for the FRT and DFRT nanoparticles shown in Figure 1e indicate significant loading of IR820 dye molecules. Next we investigated the photophysical and photochemical properties of DFRT using optical absorption and fluorescence spectra. Figure 2a and Figure S1 (Supporting Information) show UV-vis absorbance spectra of FRT, IR820 dye, and DFRT. The IR820 dye exhibits a strong peak at 687 nm, and a weak shoulder at 825 nm. FRT without dye shows virtually no absorption in the range 500–900 nm. After the dye is loaded into FRT, DFRT exhibits a weak shoulder at 761 nm, and a strong peak at 835 nm. The remarkably red-shifted absorption may be attributed to the strong interaction between the dye and FRT. The loading process is accompanied by a color
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COMMUNICATION Figure 2. Characterization of DFRT. a) UV-vis absorbance spectra of FRT, IR820 dye, and DFRT. b) Fluorescence emission spectra of IR820 dye and DFRT. c) Temperature elevation of aqueous solution samples with the same concentration as a function of irradiation time. d) Photoacoustic (PA) spectrum of DFRT.
change of the solution from dark green to brown (Figure S2a, Supporting Information). The absorption spectrum of DFRT is different from that of unloaded IR820 dye (Figure S2b–d). With different mole ratios of ferritin and IR820, that is, 1:50, 1:80, and 1:100, the IR820 loading efficiencies were 17.32, 25.11, and 29.53 wt%, respectively. We chose a mole ratio of 1:50 for the remaining experiments. The fluorescence excitation spectrum of IR820 dye shows the two excitation wavelengths, at 550 and 770 nm (Figure S3, Supporting Information). As shown in Figure 2b, both IR820 dye and DFRT exhibited a shift in emission peak from 604 nm to 834 nm as the excitation wavelength was changed from 550 nm to 770 nm. The results also show fluorescence quenching, most likely owing to the tight packing of IR820 dye molecules after being loaded into FRT. The quenching efficiency of IR820 dye in DFRT is ca. 20% upon 550 nm excitation, but almost ca. 80% upon 770 nm excitation. Based on these results, we selected 550 nm for fluorescence imaging with high fluorescence QY and 808 nm for PTT with high photothermal conversion efficiency. When separate excitation wavelengths are used, it is easy to alternate between the dual roles of DFRT — contrast agent and PTCA — for imaging and PTT. To further verify the red-shifted absorption and fluorescence quenching of dye loaded FRT, we chose ICG, a FDA-approved NIR dye, for comparison. Similar results were observed, as shown in Figure S4 (Supporting Information). ICG has a strong peak at 683 nm, and a weak shoulder at 780 nm. After being
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encapsulated into FRT, ICG-FRT exhibits a weak shoulder at 725 nm, and a strong peak at 800 nm. The loading process is also accompanied by a color change of the solution from light green to black (Figure S2). A marked fluorescence quenching of ICG upon 760 nm excitation was found after FRT loading. Interestingly, there is ca. 4.5-fold fluorescence enhancement of ICG at the 700 nm emission peak upon 630 nm excitation in the ICG-FRT system. Therefore, ICG-FRT is also suitable for simultaneous optimization of imaging and PTT using separate wavelengths, 630 nm for fluorescence imaging with high fluorescence QY and 808 nm for PAI and PTT with high photothermal conversion efficiency. These results suggest the great potential of FRT as a new type of carrier for dye delivery, fluorescence, and PAI, and enhanced PTT treatment. We next studied the potential of DFRT in PTT of cancer by measuring the temperature elevation of FRT, IR820 dye, and DFRT in aqueous solutions exposed to a 1 W cm–2 808 nm laser for 5 min (Figure 2c). No obvious temperature change was observed in the control group of pure FRT, owing to the lack of absorption of FRT in the range 500–900 nm. Upon the same laser exposure, IR820 dye and DFRT (80 µg mL–1 of dye) exhibited a temperature rise of 18.34 and 28.38 °C, respectively. These results suggest significantly higher photothermal conversion efficiency of DFRT compared to the IR820 dye upon 808 nm excitation. The effective photothermal conversion also supports the use of DFRT for PAI[15] (Figure 2d). The best excitation wavelength range is from 800 to 850 nm. To achieve
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Figure 3. In vitro cell experiments. a) Fluorescence images of calcein AM / PI co-stained 4T1 cells after incubation for 6 h with free IR820 dye or DFRT (50 µg mL–1 dye) after being exposed to 808 nm laser at different power densities of 0.5 and 1 W cm–2 for 5 min. Cells incubated with the same concentration of free IR820 dye or DFRT without laser irradiation were chosen as controls. b) Viability of 4T1 cells after being incubated with various concentrations of DFRT for 24 h. c) Viabilities of 4T1 cells after DFRT-induced PTT at different laser power densities. Cell viability was normalized to the control group without any treatment. Error bars are based on the standard deviations of five parallel samples.
in vivo simultaneous PAI and PTT, we chose 808 nm as the excitation wavelength for PAI. Encouraged by high photothermal conversion efficiency of DFRT upon 808 nm excitation, we next investigated the in vitro PTT efficacy and cytotoxicity of DFRT. Live/dead cells were differentiated by calcein AM (live cells, green fluorescence) and propidium iodide (PI) (dead cells, red fluorescence) co-staining after PTT treatment (Figure 3a). In the control groups (IR820 dye or DFRT only), all the cells displayed green fluorescence, so IR820 dye or DFRT has no measurable cytotoxicity. Upon laser irradiation at different power densities, both IR820 dye and DFRT show a laser dose–dependent PTT effect on 4T1 cells. All cells were killed after being incubated with DFRT (50 µg mL–1 of IR820 dye) and exposed to the NIR laser at 1 W cm–2 for 5 min. As shown in Figure S5 (Supporting Information), on the boundary of the laser spot, only cells within the laser spot were killed, showing intense homogeneous red fluorescence. The cells outside the region of the laser spot stayed alive, showing strong green fluorescence. To further evaluate the cytotoxicity and PTT efficacy of DFRT, the viability of 4T1 cells after being exposed to various concentrations of DFRT for 24 h without laser irradiation were assessed (Figure 3b). We found DFRT exhibited negligible toxicity at all the test concentrations. Even with 400 µg mL–1 of DFRT, the cell viability is over 90%, which suggests that DFRT alone has negligible adverse effect on tumor cells. To verify the improved PTT efficacy induced by DFRT, three groups — with FRT, IR820 dye, or DFRT — were compared under the same conditions with increasing laser power. For the FRT group, no PTT effect was observed at any of the studied laser power densities. Upon laser irradiation, both IR820 dye and DFRT
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induced a laser dose–dependent cytotoxicity to 4T1 cells, which was significantly different from that of control groups as shown in Figure 3c. DFRT exhibited a much higher PTT efficacy than IR820 dye under the same conditions, which is attributed to the higher photothermal conversion efficiency of DFRT than IR820 dye. The above results are in good agreement with live/dead cell staining results. To investigate the stability of DFRT at the cellular level, we prepared fluoroscein isothiocyanate (FITC)-conjugated cyanine5.5 (Cy5.5)-loaded FRT (FITC-FRT-Cy5.5). FITC was covalently coupled to primary amines of FRT. FRT was loaded with Cy5.5 by the same method. After 6 h incubation of FITC-FRTCy5.5 with 4T1 cells, we used LysoTracker (a fluorescent dye probe) to stain lysosomes and observed FITC-FRT-Cy5.5 using confocal laser scanning microscopy (CLSM). Figure S6 (Supporting Information) clearly shows that the blue fluorescence signals from LysoTracker match well with the fluorescence signals from FITC-FRT (green) and Cy5.5 (red), suggesting FITCFRT and Cy5.5 colocalize within the lysosomes. These results indicate FITC-FRT-Cy5.5 is stable in lysosomes during the 6 h incubation. It would be reasonable to believe that DFRT is also stable in lysosomes. We next investigated the feasibility of using DFRT for in vivo multimodal imaging-guided PTT. To achieve the simultaneous optimization of NIR fluorescence and PAI, we used two separate wavelengths to excite DFRT: 550 nm for fluorescence imaging with high fluorescence QY and 808 nm for PAI with high photothermal conversion efficiency. Nude mice with subcutaneous 4T1 tumors were selected as the animal model. When the tumors reached 60 mm3, the mice were treated with an intravenous injection of DFRT (2 mg mL–1 of IR820 dye,
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COMMUNICATION Figure 4. In vivo NIR fluorescence (FL) and photoacoustic (PA) imaging. a)In vivo NIR FL images in mice taken at different times after intravenous injection of DFRT. b) Ex vivo NIR FL images of mouse tissues (from left to right: heart, liver, spleen, lung, kidneys, tumor), harvested 24 h after injection of free IR820 dye or DFRT. c)In vivo 2D and 3D cross-sectional US and PA images of tumor tissues in mice taken at different times after intravenous injection of free IR820 dye or DFRT. d) Change of FL signal of tumors with time following intravenous injection of DFRT. e) Biodistribution of free IR820 dye or DFRT in 4T1 tumor-bearing nude mice 24 h after injection. f) Change of PA signal of tumors with time following intravenous injection of free IR820 dye or DFRT. For all plots: mean ± standard deviation; n = 3 per group
200 µL). NIR fluorescence imaging was employed to monitor the pharmacokinetics of DFRT in vivo. As shown in Figure 4a, distributed DFRT fluorescence was found over the whole body soon after injection. With time, the whole-body signal gradually decreased and the tumor signal increased. The tumor can be easily differentiated from the surrounding normal tissue with good contrast 4–24 h post-injection. After 24 h, the major organs of mice injected with unloaded dye or DFRT were harvested for ex vivo imaging (Figure 4b). Meanwhile, PAI was also used to observe the PA signal change in tumor tissues. Figure 4c shows snapshots of mice taken at various times of in vivo two-dimensional (2D) and 3D cross-sectional ultrasonic (US) and PA images upon 808 nm excitation of the tumor area after intravenous injection of IR820 dye or DFRT. A gradual increase of PA signal was observed in the tumor regions of DFRT-treated mice. DFRT accumulation in the tumor was quantified by dye fluorescence intensity (Figure 4d). The average fluorescence intensity from dye accumulated in the tumor areas quickly rose in the period 1–4 h after injection and reached a plateau 4–24 h after injection, followed by a slight decrease over time. Meanwhile, the quantification of tissue/organ distribution was carried out by means of ex vivo imaging (Figure 4e). The relative
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uptake efficiency (10.48 ± 0.93%) of DFRT is ca. four times that of IR820 dye (2.64 ± 0.20%), which suggests that the FRT carrier significantly improved the tumor accumulation rate of the dye. The strong fluorescence signals in the liver and spleen may be attributed to the macrophage clearance of IR820 dye and DFRT from the blood. Strong kidney signals imply renal clearance of IR820 dye and DFRT. Semi-quantification of PA signals in the tumor area revealed that the average tumor PA intensity (0.95 ± 0.01 a.u.) of DFRT was ca. 2.6 times that of IR820 dye (0.37 ± 0.02 a.u.) (Figure 4f), which further confirms that the FRT carrier can significantly improve tumor accumulation of the dye molecules. The tumor tissue turned a dark color with increased accumulation of DFRT at long times after intravenous injection (Figure S7, Supporting Information). Based on the effective DFRT-induced PTT in vitro and the high tumor accumulation rate of DFRT in vivo, in vivo PTT studies were carried out on the same animal model. According to the observations of fluorescence and PA imaging, the most suitable time to implement PTT is 24 h after injection of DFRT. Upon 808 nm laser irradiation, thermal imaging was employed to monitor the temperature change in vivo using an infrared (IR) thermal camera (Figure 5a,c). For mice injected with DFRT, the tumor temperature changes were 23.8 or 42.2 °C
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Figure 5. In vivo PTT. a) Thermal images of 4T1 tumor-bearing mice after injection of free IR820 dye or DFRT and exposure to 808 nm laser irradiation. b) 3D color Doppler images of tumor before and 12 h after PTT treatment. c) Heating curves of 4T1 tumors upon laser irradiation as a function of irradiation time. d) Tumor growth curves of different groups of 4T1 tumor-bearing mice after treatment. Tumor volumes have been normalized to their initial sizes. Error bars represent the standard deviations of 4–6 mice per group. * P < 0.01. e) Survival curves of tumor-bearing mice after various treatments. DFRT-injected mice, after PTT treatment, showed 100% survival over 40 days. f) H&E staining of tumor sections collected from different groups of mice 2 h after laser irradiation.
within 10 min, upon 0.5 or 1 W cm–2 808 nm laser irradiation, respectively. No significant temperature rise was observed in other body parts of the mice (Figure S8, Supporting Information). In contrast, upon 1 W cm–2 808 nm laser irradiation, the local temperature of tumors treated with phosphate-buffered saline (PBS) or IR820 dye increased by about 7.2 or 11.1 °C, respectively. Figure 5b shows 3D color Doppler images of a tumor before and after PTT treatment. While the tumor clearly displayed blood flow prior to treatment, no blood flow was observed in the tumor after treatment, which can be attributed to the destruction of the blood vessels of tumor tissue during treatment. For in vivo DFRT-induced PTT treatment, eight groups of 4T1 tumor-bearing mice (4–6 mice per group) were designated
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as follows: untreated mice (control, n = 6), mice with PBS administration and laser irradiation (PBS+laser, n = 6), mice with FRT administration but no laser irradiation (FRT, n = 4), mice with FRT administration and laser irradiation (FRT+laser, n = 4), mice with dye administration but no laser irradiation (IR820 dye, n = 4), mice with dye administration and laser irradiation (IR820 dye+laser, n = 5), mice with DFRT administration but no laser irradiation (DFRT, n = 4), and mice with DFRT administration and laser irradiation (DFRT+laser, n = 6). The same 808 nm laser density (0.5 W cm–2) and exposure time (10 min) were applied in all eight treatments. While the tumors in the control groups all grew at similar speeds, the tumors in both the IR820 dye+laser and DFRT+laser groups exhibited remarkable delay in tumor growth or tumor regression after
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and 808 nm for PAI and PTT with high photothermal conversion efficiency, we achieved optimization of both imaging and PTT in our system. The cancer theranostic capability of DFRT was carefully investigated both in vitro and in vivo. Most intriguingly, 100% in vivo tumor elimination is produced by intravenous injection of DFRT, under a low laser power density of 0.5 W cm–2 without weight loss, tumor recurrence, and noticeable toxicity. This concept of protein-based theranostics has potential applications for clinical translation of personalized nanomedicine with targeted dye/drug delivery, diagnosis, and treatment.
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two weeks (IR820 dye+laser vs. ctrl, P < 0.05; DFRT+laser vs. ctrl, P < 0.001) (Figure 5d). In the DFRT+laser group, all the tumors were effectively ablated, leaving scars at their original sites, without showing recurrence (Figure S9, Supporting Information). Additionally, it is also worth noting that the DFRT group exhibited significantly higher PTT therapeutic efficacy than the IR820 dye+laser group on day 14 (DFRT+laser vs. IR820 dye+laser, P < 0.0001). As shown in Figure 5e, DFRT administration and laser irradiation greatly prolonged mice survival over 40 days, without a single death or tumor recurrence. In contrast, for the IR820 dye+laser group, all the mice had to be sacrificed on day 20 owing to the extensive tumor burden. Mice in other control groups showed average lifespans of ca. 14 days after treatment started. Hematoxylin and eosin (H&E) stains of tumor sections were collected from four laser irradiation groups of mice 2 h after laser irradiation (Figure 5f). For the PBS+laser and FRT+laser groups, the histological section showed observable errhysis (minor bleeding) and the infiltration of a small number of inflammatory cells without tissue structure damage. For the IR820 dye+laser group, inflammatory cell infiltration, cell death, and errhysis with partial tissue structure damage was observed. For the DFRT+laser group, similar results were observed; however, instead of errhysis with partial tissue structure damage, serious tissue structure damage was detected. In the high-resolution H&E images (Figure S10, Supporting Information), we found significant cancer cell damage with nuclear membrane fragmentation (karyorrhexis) and nuclei shrinkage with pyknosis (condensation of chromatin) in the DFRT+laser group. The irreversible condensation of chromatin and karyorrhexis in the nuclei of tumor cells led to intensive necrosis or apoptosis. The results indicated that the in vivo PTT therapeutic efficacy of DFRT was significantly higher than that of IR820 dye. Furthermore, no significant body weight variation was noticed after DFRT-induced PTT treatment (Figure S11, Supporting Information). After DFRT administration and laser irradiation, main organs from different groups of mice, as seen through H&E stained images (Figure S12, Supporting Information), showed no obvious damage or inflammation. In accordance with the fact that DFRT has low cytotoxicity and immunogenicity, it is valid to surmise that circulating DFRT does not induce discernible toxic side effects in treated animals. Thus, owing to its excellent theranostic capability without noticeable toxicity and immunogenicity, DFRT is a suitable candidate for multimodal imaging-guided PTT in vivo. The following may be considered in future applications of DFRT for cancer theranostics: 1) control of the arrangement of dye molecules inside FRT; 2) precise control of the size of DFRT; 3) enhanced knowledge of the stability of DFRT in vivo; 4) modification of the surface of FRT with specific biomarkers to further improve the tumor accumulation rate of DFRT; 5) systematic assessment of the toxicity, biocompability, immunogenicity, pharmacokinetics, and biodistribution of DFRT in vivo. In summary, we have developed a ferritin (DFRT) nanocage, loaded with the near-infrared dye IR820, that shows strong absorbance in the NIR region for simultaneous fluorescence/ photoacoustic/photothermal multimodal imaging-guided enhanced PTT. Using two different wavelengths for excitation, 550 nm for fluorescence imaging with high fluorescence QY
Supporting Information Supporting Information is available from the Wiley Online Library or from the authors.
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